Analogs of parathyroid hormone and parathyroid hormone related peptide: synthesis and use for the treatment of osteoporosis

ABSTRACT

Synthetic polypeptide analogs of parathyroid hormone PTH, parathyroid hormone related peptide PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, in which amino acid residues (22-31) form an amphipathic α-helix, these residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence: 
     
         Haa (Laa Laa Haa Haa).sub.2 Laa 
    
     and their pharmaceutically acceptable salts are useful for the prophylaxis and treatment of ostoporosis in mammals. Processes for the production of the polypeptides via solid phase and recombinant methods are provided.

This is a division of application Ser. No. 07/915,247 filed Jul. 14, 1992, now U.S. Pat. No. 5,589,452.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel analogs of parathyroid hormone and parathyroid hormone related peptide, their synthesis by solid phase and recombinant techniques, and their use for increasing bone mass in mammalian subjects.

2. Description of Related Art

Osteoporosis is the most common form of metabolic bone disease and may be considered the symptomatic, fracture stage of bone loss (osteopenia). Although osteoporosis may occur secondary to a number of underlying diseases, 90% of all cases appear to be idiopathic. Postmenopausal women are particularly at risk for idiopathic osteoporosis (postmenopausal or Type I osteoporosis). Another high risk group for idiopathic osteoporosis is the elderly of either sex (senile or Type II osteoporosis). Osteoporosis has also been related to corticosteroid use, immobilization or extended bed rest, alcoholism, diabetes, gonadotoxic chemotherapy, hyperprolactinemia, anorexia nervosa, primary and secondary amenorrhea, and oophorectomy.

In the various forms of osteoporosis, bone fractures, which are the result of bone loss that has reached the point of mechanical failure, frequently occur. Postmenopausal osteoporosis is characterized by fractures of the wrist and spine, while femoral neck fractures seem to be the dominant feature of senile osteoporosis.

The mechanism by which bone is lost in osteoporotics is believed to involve an imbalance in the process by which the skeleton renews itself. This process has been termed bone remodeling. It occurs in a series of discrete pockets of activity. These pockets appear spontaneously within the bone matrix on a given bone surface as a site of bone resorption. Osteoclasts (bone dissolving or resorbing cells) are responsible for the resorption of a portion of bone of generally constant dimension. This resorption process is followed by the appearance of osteoblasts (bone forming cells) which then refill with new bone the cavity left by the osteoclasts.

In a healthy adult subject, the rate at which osteoclasts and osteoblasts are formed is such that bone formation and bone resorption are in balance. However, in osteoporotics an imbalance in the bone remodeling process develops which results in bone being lost at a rate faster than it is being made. Although this imbalance occurs to some extent in most individuals as they age, it is much more severe and occurs at a younger age in postmenopausal osteoporotics or following oophorectomy.

There have been many attempts to treat osteoporosis with the goal of either slowing further bone loss or, more desirably, producing a net gain in bone mass. Certain agents, such as estrogen and the bisphosphonates, appear to slow further bone loss in osteoporotics. Agents which slow bone loss, because of the different durations of bone resorption and formation, may appear to increase bone mass (on the order of 3 to 7%). However, this apparent increase is limited in time, not progressive, and is due to a decrease in "remodeling space." In addition, because of the close coupling between resorption and formation, treatments which impede bone resorption also ultimately impede bone formation.

It has been suggested that treatment with parathyroid hormone (PTH) would lead to both increased bone turnover and a positive calcium balance. However, human clinical trials have shown that any increase in trabecular bone is offset by a decrease in cortical bone, so that there is no net increase in total bone.

Hefti, et al. in Clinical Science 62, 389-396 (1982) have reported that daily subcutaneous doses of either bPTH(1-84) or hPTH(1-34) increased whole body calcium and ash weight of individual bones in both normal and osteoporotic adult female rats.

Liu, et al. in J. Bone Miner. Res. 6:10, 1071-1080 (1991) have noted that ovariectomy of adult female rats induced a 47% loss in the percentage of trabecular bone in the proximal tibial metaphysis, accompanied by a significant increase in the number of osteoblasts and trabecular osteoclasts. Daily subcutaneous injections of hPTH(1-34) completely reversed the loss of trabecular bone and resulted in amounts of trabecular bone exceeding that of sham operated controls. The number of osteoblasts increased and the number of osteoclasts decreased.

Hock et al. in J. Bone Min. Res. 7:1, 65-71 (1992) have reported that daily subcutaneous injections of hPTH(1-34) to healthy adult male rats for 12 days increased trabecular and cortical bone calcium and dry weight. Total bone mass, trabecular bone volume, trabecular thickness and number, and osteoblastic surfaces were increased.

The mammalian parathyroid hormones, e.g. human (hPTH), bovine (bPTH), and porcine (pPTH), are single polypeptide chains of 84 amino acid residues, with molecular weights of approximately 9500. Biological activity is associated with the N-terminal portion, with residues (1-34) apparently the minimum required.

The N-terminal segment of human PTH differs from the N-terminal segment of the bovine and porcine hormones by only three and two amino acid residues, respectively: ##STR1##

The primary function of PTH is to elicit the adaptive changes that serve to maintain a constant concentration of Ca²⁺ in the extracellular fluid. PTH acts on the kidneys to increase tubular reabsorption of Ca²⁺ from the urine, as well as stimulating the conversion of calcifediol to calcitriol, which is responsible for absorption of Ca²⁺ from the intestines. One prominent effect is to promote the mobilization of Ca²⁺ from bone. PTH acts on bone to increase the rate of resorption of Ca²⁺ and phosphate. PTH stimulates the rate of bone resorption by osteoclasts, increases the rate of differentiation of mesenchymal cells to osteoclasts, and prolongs the half life of these latter cells. With prolonged action of PTH the number of bone forming osteoblasts is also increased; thus, the rate of bone turnover and remodeling is enhanced. However, individual osteoblasts appear to be less active than normal.

Rosenblatt, et al. in U.S. Pat. Nos. 4,423,037, 4,968,669 and 5,001,223 have disclosed PTH antagonists obtained by the deletion of the N-terminal (1-6) amino acids and the selective replacement of Phe⁷, Met⁸,18, and Gly¹². Tyr³⁴ --NH₂ reportedly increased the activity and stability of these compounds.

Parathyroid hormone-related peptide (PTHrp), a 140+ amino acid protein, and fragments thereof, reproduce the major biological actions of PTH. PTHrp is elaborated by a number of human and animal tumors and other tissues and may play a role in hypercalcemia of malignancy. The sequence of hPTHrp (1-34) is as follows: ##STR2##

The sequence homology between hPTH and hPTHrp is largely limited to the 13 N-terminal residues, 8 of which are identical; only 1 of 10 amino acids in the (25-34) receptor binding region of hPTH is conserved in hPTHrp. Conformational similarity may underlie the common activity. Cohen, et al. in J. Biol. Chem. 266:3, 1997-2004 (1991) have suggested that much of the sequence of PTH(1-34) and PTHrp(1-34), in particular regions (5-18) and (21-34), assumes an α-helical configuration, while noting that there is some question whether this configuration prevails for the carboxyl terminal end under physiological conditions. Such a secondary structure may be important for lipid interaction, receptor interaction, and/or structural stabilization.

We have synthesized analogs of PTH and of PTHrp with the objective of developing improved therapeutic agents for the restoration of bone mass in mammalian subjects, including those afflicted with osteoporosis.

SUMMARY OF THE INVENTION

This invention provides synthetic polypeptide analogs of parathyroid hormone (PTH), parathyroid hormone related peptide (PTHrp), and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

When specific illustrative embodiments of this sequence type are inserted into PTH, PTHrp, and the physiologically active truncated analogs and homologs of PTH and PTHrp, the resulting polypeptides are effective bone remodeling agents.

In one aspect, then, this invention provides analogs of PTH, PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, or salts thereof, in which amino acid residues (22-31) form an amphipathic α-helix, the sequence of said residues (22-31) selected from: ##STR3## wherein Xaa is Glu or Arg (SEQ ID NO:26); ##STR4## wherein Xaa²⁹ is Glu, Lys, or Lys(COCH₂ PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ ID NO:27); ##STR5##

Also provided are pharmaceutical compositions for the prevention or treatment of conditions characterized by decreases in bone mass comprising an effective bone mass increasing amount of a polypeptide analog of parathyroid hormone (PTH), parathyroid hormone related peptide (PTHrp), and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

Further, this invention provides methods for treating mammalian conditions characterized by decreases in bone mass which methods comprise administering to a mammal in need thereof an effective bone mass increasing amount of a polypeptide analog of PTH, PTHrp, or of a physiologically active truncated homolog or analog of PTH or PTHrp, or a salt thereof, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

More specifically, this invention provides methods for treating mammalian conditions characterized by decreases in bone mass which methods comprise administering to a mammal in need thereof an effective bone mass increasing amount of a polypeptide analog of PTH, PTHrp, or of a physiologically active truncated homolog or analog of PTH or PTHrp, or salt thereof, in which amino acid residues (22-31) form an amphipathic α-helix, the sequence of said residues (22-31) selected from: (SEQ ID NOS: 26, 27, 28, 29, and 30).

This invention also includes processes for the solid phase synthesis of polypeptide analogs of PTH, PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa,

which processes comprise sequentially coupling protected amino acids on a suitable resin support, removing the side chain and N.sup.α -protecting groups, and cleaving the polypeptide from the resin.

This invention also encompasses processes for the solid phase synthesis of polypeptide analogs of PTH, pTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic α-helix, the sequence of said residues (22-31) selected from: (SEQ ID NOS: 26, 27, 28, 29, and 30); which processes comprise sequentially coupling protected amino acids on a suitable resin support, removing the side chain and N.sup.α -protecting groups, and cleaving the polypeptide from the resin. Also included are DNA sequences, vectors, and plasmids for the recombinant synthesis of polypeptide analogs of PTH, PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

In another aspect this invention provides DNA sequences, vectors, and plasmids for the recombinant synthesis of polypeptide analogs of PTH, PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic α-helix, the sequence of said residues (22-31) selected from: (SEQ ID NOS: 26, 27, 28, 29, and 30).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the DNA sequence and enzyme restriction sites of a synthetic gene coding for a PTHrp(1-34) analog of this invention.

FIG. 2 outlines the preparation of a plasmid incorporating a PTHrp(1-34) analog gene.

FIG. 3 outlines the preparation of a plasmid incorporating two copies of a PTHrp(1-34) analog gene.

FIG. 4 outlines the preparation of a plasmid incorporating four copies of a PTHrp(1-34) analog gene.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

The one- and three-letter abbreviations for the various common nucleotide bases and amino acids are as recommended in Pure Appl. Chem. 31, 639-645 (1972) and 40, 277-290 (1974) and comply with 37 CFR §1.822 (55 FR 18245, May 1, 1990). The abbreviations represent L-amino acids unless otherwise designated as D- or D,L-. Certain amino acids, both natural and non-natural, are achiral, e.g. glycine. All peptide sequences are presented with the N-terminal amino acid on the left and the C-terminal amino acid on the right.

Further abbreviations for other amino acids and compounds used herein are:

    ______________________________________                                         hSer        homoserine                                                         hSerlac     homoserine lactone                                                 Nle         norleucine                                                         PEG2        radical of diethylene glycol methyl                                            ether, a.k.a. methoxydi(ethyleneoxy),                                          CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.2 --, (MW = 119)               PEG5000     radical of poly(ethylene glycol methyl                                         ether), a.k.a. methoxypoly(ethyleneoxy),                                       CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.110 --, (avg. MW = 5000)       PEGX        radical of poly(ethylene glycol methyl                                         ether), CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.n -, n = 2-225,                    (avg. MW = 100 to 10,000)                                          ______________________________________                                    

"Hydrophilic amino acid (Haa)" refers to an amino acid having at least one hydrophilic functional group in addition to those required for peptide bond formation, such as arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, and their homologs.

"Lipophilic amino acid (Laa)" refers to an uncharged, aliphatic or aromatic amino acid, such as isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine, and their homologs.

For the purposes of this invention, alanine is classified as "amphiphilic" i.e., capable of acting as either hydrophilic or lipophilic.

"Physiologically active truncated homolog or analog of PTH or PTHrp" refers to a polypeptide having a sequence comprising less than the full complement of amino acids found in PTH or PTHrp which, however, elicits a similar physiological response. The truncated PTH or PTHrp need not be fully homologous with PTH or PTHrp to elicit a similar physiological response. PTH(1-34) and PTHrp(1-34) are preferred, but not exclusive, representatives of this group.

"Amphipathic α-helix" refers to the secondary structure exhibited by certain polypeptides in which the amino acids assume an α-helical configuration having opposing polar and nonpolar faces oriented along the long axis of the helix. The possibility of α-helical structure in the polypeptide of interest may be explored to some extent by the construction of a "Schiffer-Edmundson wheel" (M. Schiffer and A. B. Edmundson, Biophys. J. 7, 121 (1967), incorporated herein by reference), of the appropriate pitch and noting the segregation of the hydrophilic and lipophilic residues on opposite faces of the cylinder circumscribing the helix. Alternatively, empirical evidence, such as circular dichroism or x-ray diffraction data, may be available indicating the presence of an α-helical region in a given polypeptide. An ideal α-helix has 3.6 amino acid residues per turn with adjacent side chains separated by 100° of arc.

Eisenberg et al. in Nature 299:371-374 (1982) and Proc. Nat. Acad. Sci. USA 81:140-144 (1984) have combined a hydrophobicity scale with the helical wheel to quantify the concept of amphipathic helices. The mean hydrophobic moment is defined as the vector sum of the hydrophobicities of the component amino acids making up the helix. The following hydrophobicities for the amino acids are those reported by Eisenberg (1984) as the "consensus" scale:

Ile 0.73; Phe 0.61; Val 0.54; Leu 0.53; Trp 0.37;

Met 0.26 Ala 0.25; Gly 0.16; Cys 0.04; Tyr 0.02;

Pro -0.07; Thr -0.18; Ser -0.26; His -0.40; Glu -0.62;

Ash -0.64; Gln -0.69; Asp -0.72; Lys -1.10; Arg -1.76.

The hydrophobic moment, μ_(H), for an ideal α-helix having 3.6 residues per turn (or a 100° arc (=360°/3.6) between side chains), may be calculated from:

    μ.sub.H = (Σ H.sub.N sinδ(N-1)).sup.2 +(Σ H.sub.N cosδ(N-1)).sup.2 !.sup.1/2,

where H_(N) is the hydrophobicity value of the N^(th) amino acid and the sums are taken over the N amino acids in the sequence with periodicity δ=100°. The hydrophobic moment may be expressed as the mean hydrophobic moment per residue by dividing μ_(H) by N to obtain <μ_(H) >. A value of <μ_(H) > at 100°±20° of about 0.20 or greater is suggestive of amphipathic helix formation. The <μ_(H) > values at 100° for hPTHrp (22-31) and hPTH (22-31) are 0.19 and 0.37, respectively.

Cornett, et al., in J. Mol. Biol., 195:659-685 (1987) have further extended the study of amphiphathic α-helices by introducing the "amphipathic index" as a predictor of amphipathicity. They concluded that approximately half of all known α-helices are amphipathic, and that the dominant frequency is 97.5° rather than 100°, with the number of residues per turn being closer to 3.7 than 3.6. While such refinements are scientifically interesting, the basic approach of Eisenberg, et al. is sufficient to classify a given sequence as amphipathic, particularly when one is designing a sequence ab initio to form an amphipathic α-helix.

A substitute amphipathic α-helical amino acid sequence may lack homology with the sequence of a given segment of a naturally occurring polypeptide but elicits a similar secondary structure, i.e. an α-helix having opposing polar and nonpolar faces, in the physiological environment. Replacement of the naturally occurring amino acid sequence with an alternative sequence may beneficially affect the physiological activity, stability, or other properties of the altered parent polypeptide. Guidance as to the design and selection of such sequences is provided in J. L. Krstenansky, et al., FEBS Letters 242:2, 409-413 (1989), and J. P. Segrest, et al. Proteins: Structure, Function, and Genetics 8:103-117 (1990) among others.

The ten amino acid amphipathic α-helix of this invention has the formula:

    Haa (Laa Laa Haa Haa).sub.2 Laa

wherein the Haa's are selected from the group of hydrophilic amino acids and the Laa's are selected from the group of lipophilic amino acids, as defined above. Assuming an idealized α-helix, residues 1, 4, 5, 8, and 9 are distributed along one face (A) of the helix within about a 140° arc of each other, while residues 2, 3, 6, 7, and 10 occupy an opposing 140° arc on the other face (B) of the helix. Preferably, all the residues on one face are of the same polarity while all those on the other face are of the opposite polarity, i.e., if face A is all hydrophilic, face B is all lipophilic and vice versa. The skilled artisan will recognize that while the helices of this invention are described by

    Haa(Laa Laa Haa Haa).sub.2 Laa,

the reverse sequence,

    Laa (Haa Haa Laa Laa).sub.2 Haa

will also meet the residue distribution criteria and is an equivalent descriptor of the helices of this invention.

Alanine may be substituted for either hydrophilic or lipophilic amino acids, since Ala can reside readily on either face of an amphipathic α-helix, although Ala₁₀ does not form an amphipathic α-helix. Generally, proline, cysteine, and tyrosine are not used; however, their presence and other random errors in the sequence may be tolerated, e.g. a hydrophilic residue on the lipophilic face, as long as the remaining amino acids in the segment substantially conform to the hydrophilic face-lipophilic face division. A convenient method for determining if a sequence is sufficiently amphipathic to be a sequence of this invention is to calculate the mean hydrophobic moment, as defined above. If the peak mean moment per residue at 100°±20° exceeds about 0.20, then the sequence will form an amphipathic helix and is a sequence of this invention.

For example, the mean hydrophobic moment per residue at 100° for (SEQ ID NO: 26), Xaa=Glu, is calculated as follows:

    ______________________________________                                         A.A.    H.sub.N                                                                               δ (N-1)                                                                            H sin δ (N-1)                                                                     H cos δ (N-1)                          ______________________________________                                         E       -.62    0        0        -.62                                         L       .53    100       .52      -.17                                         L       .53    200       -.18     -.50                                         E       -.62   300       .34      -.31                                         K       -1.1   400       -.70     -.85                                         L       .53    500       .34      -.41                                         L       .53    600       -.46     -.27                                         E       -.62   700       .21      -.58                                         K       -1.1   800       -1.08    -.19                                         L       .53    900       0        -.53                                                                  Σ = 0.81                                                                          Σ = -4.43                              ______________________________________                                    

    μ.sub.H = (0.81).sup.2 +(-4.43).sup.2 !.sup.1/2 =4.50

    <μ.sub.H >=4.50/10=0.45

For this sequence, the mean peak hydrophobic moment occurs at 92° and has a value of 0.48.

In applying this concept to parathyroid hormone and parathyroid hormone related peptide, it was hypothesized that either or both regions (7-16) and (22-31) may exhibit α-helical secondary structure and could be replaced with a non-homologous sequence having similar structural tendencies, without loss of biological activity or induction of immunoreaction.

Preferred Embodiments

In one aspect this invention includes polypeptide analogs of the physiologically active truncated homolog hPTHrp(1-34), as shown in Formula (I):

    Ala Val Ser Glu Xaa.sup.5 Gln Leu Leu His Asp Xaa.sup.11 Gly Xaa.sup.13 Ser Ile Gln Asp Leu Xaa.sup.19 Arg Xaa.sup.21 Xaa.sup.22-31 Xaa.sup.32 Xaa.sup.33 Xaa.sup.34 Term,

wherein:

Xaa⁵ is His or Ala;

Xaa¹¹ and Xaa¹³ are independently Lys, Arg, or Leu;

Xaa¹⁹ and Xaa²¹ are independently Ala or Arg;

Xaa²²⁻³¹ is selected from: ##STR6## wherein Xaa is Glu or Arg (SEQ ID NO:26); ##STR7## wherein Xaa is Glu, Lys, or Lys(COCH₂ PEGX) and PEGX is a poly(ethylene glycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ ID NO:27); ##STR8## Xaa³² is His or Lys; Xaa³³ is Thr, Glu, or Ala;

Xaa³⁴ is Ala, hSer, Tyr, or Leu;

and Term is Gly Arg Arg, lactone, OH or NR₂, where each R is H or (C₁ -C₄) alkyl; and their pharmaceutically acceptable salts. (Formula I)

A more specific aspect of the invention includes those polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26), for which <μ_(H) > at 100° exceeds 0.45. A still more specific aspect of the invention includes those Formula (I) polypeptides wherein Xaa²²⁻³¹ is (SEQ ID NO:26); Xaa¹¹ and Xaa¹³ are both Lys; and Xaa¹⁹ and Xaa²¹ are both Arg. Representative polypeptides include, but are not limited to: ##STR9##

Another aspect of this invention includes those polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26); Xaa¹¹ and Xaa¹³ are both Lys; and one of Xaa¹⁹ and Xaa²¹ is Arg and the other is Ala. Representative poly-peptides of this subgenus include, but are not limited to: ##STR10##

In another aspect this invention includes those polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26); one of Xaa¹¹ and Xaa¹³ is Leu and the other is Lys; and Xaa¹⁹ and Xaa²¹ are both Arg. Representative poly-peptides of this subgenus include, but are not limited to: ##STR11##

In another aspect this invention includes those polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:27), for which <μ_(H) > at 100° exceeds 0.50. A further aspect of this invention includes those Formula (I) polypeptides wherein Xaa²²⁻³¹ is (SEQ ID NO:27); Xaa¹¹ and Xaa¹³ are both Lys or both Arg; and Xaa¹⁹ and Xaa²¹ are both Arg. Representative polypeptides of this subgenus include, but are not limited to: ##STR12##

In another aspect this invention includes polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:28), for which <μ_(H) > at 100° is about 0.25. Representative polypeptides of this subgenus include, but are not limited to: ##STR13##

In another aspect this invention includes polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:29), for which <μ_(H) > at 100° is about 0.28. Representative polypeptides of this subgenus include, but are not limited to: ##STR14##

In another aspect this invention includes polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:30), for which <μ_(H) > at 100° is about 0.29. Representative polypeptides of this subgenus include, but are not limited to: ##STR15##

Still another aspect of this invention includes polypeptide analogs of the physiologically active truncated homolog bPTH(1-34), as shown in Formula (II):

    Xaa.sup.1 Val Ser Glu Ile Gln Xaa.sup.7 Xaa.sup.8 His Asn Leu Gly Lys His Leu Xaa.sup.16 Ser Xaa.sup.18 Xaa.sup.19 Arg Xaa.sup.21 Xaa.sup.22-31 His Asn Xaa.sup.34 Term,

wherein:

Xaa¹ is Ser or Ala;

Xaa⁷ is Leu or Phe;

Xaa⁸ is Met or Nle;

Xaa¹⁶ is Asn or Ser;

Xaa¹⁸ is Leu, Met, or Nle;

Xaa¹⁹ is Glu or Arg;

Xaa²¹ is Val or Arg;

Xaa²²⁻³¹ is selected from (SEQ ID NO:26, 27, 28, 29, and 30);

Xaa³⁴ is Phe or Tyr;

Term is OH or NR₂, where each R is H or (C₁ -C₄)alkyl; and the pharmaceutically acceptable salts thereof. (Formula II) Representative polypeptides include, but are not limited to: ##STR16##

The skilled artisan will appreciate that numerous permutations of the polypeptide analogs may be synthesized which will possess the desirable attributes of those described herein provided that an amino acid sequence having a mean hydrophobic moment per residue at 100°±20° greater than about 0.20 is inserted at positions (22-31).

Classical Synthesis of the Polypeptides

The polypeptides of the instant invention may be synthesized by methods such as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis. The disclosures of the foregoing treatises are incorporated by reference herein.

In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.

A preferred method of preparing the analogs of the physiologically active truncated polypeptides, having fewer than about forty amino acids, involves solid phase peptide synthesis. In this method the α-amino (N.sup.α) functions and any reactive side chains are protected by acid- or base-sensitive groups. The protecting group should be stable to the conditions of peptide linkage formation, while being readily removable without affecting the extant polypeptide chain. Suitable α-amino protecting groups include, but are not limited to t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc), isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and the like, preferably t-butoxycarbonyl (Boc). Suitable side chain protecting groups include, but are not limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl, t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos), trimethylsilyl, and trityl.

In solid phase synthesis, the C-terminal amino acid is first attached to a suitable resin support. Suitable resin supports are those materials which are inert to the reagents and reaction conditions of the stepwise condensation and deprotection reactions, as well as being insoluble in the media used. Examples of commercially available resins include styrene/divinylbenzene resins modified with a reactive group, e.g., chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylated co-poly-(styrene-divinylbenzene), and the like. Benzylated, hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred. When the C-terminus of the compound is an amide, a preferred resin is p-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin.

Attachment to the PAM resin may be accomplished by reaction of the N.sup.α -protected amino acid, preferably the Boc-amino acid, as its ammonium, cesium, triethylammonium, 1,5-diazabicyclo- 5.4.0!undec-5-ene, tetramethylammonium, or similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like, preferably the cesium salt in DMF, with the resin at an elevated temperature, for example between about 40° and 60° C., preferably about 50° C., for from about 12 to 72 hours, preferably about 48 hours.

The N.sup.α -Boc-amino acid may be attached to the benzhydrylamine resin by means of, for example, an N,N'-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2 to about 24 hours, preferably about 2 hours at a temperature of between about 10° and 50° C., preferably 25° C. in a solvent such as dichloromethane or dimethylformamide, preferably dichloromethane.

The successive coupling of protected amino acids may be carried out by methods well known in the art, typically in an automated peptide synthesizer. Following neutralization with triethylamine or similar base, each protected amino acid is preferably introduced in approximately 1.5 to 2.5 fold molar excess and the coupling carried out in an inert, nonaqueous, polar solvent such as dichloromethane, DMF, or mixtures thereof, preferably in dichloromethane at ambient temperature. Representative coupling agents are N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropyl-carbodiimide (DIC) or other carbodiimide, either alone or in the presence of 1-hydroxybenzotriazole (HOBt), O-acyl ureas, benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBop), N-hydroxysuccinimide, other N-hydroxyimides, or oximes. Alternatively, protected amino acid active esters (e.g. p-nitrophenyl, pentafluorophenyl and the like) or symmetrical anhydrides may be used.

At the end of the solid phase synthesis the fully protected peptide is removed from the resin. When the linkage to the resin support is of the benzyl ester type, cleavage may be effected by means of aminolysis with an alkylamine or fluoroalkylamine for peptides with an alkylamide C-terminus, or by aminolysis with, for example, ammonia/methanol or ammonia/ethanol for peptides with an unsubstituted amide C-terminus, at a temperature between about -10° and 50° C., preferably about 25° C., for between about 12 and 24 hours, preferably about 18 hours. Peptides with a hydroxy C-terminus may be cleaved by HF or other strongly acidic deprotection regimen or by saponification. Alternatively, the peptide may be removed from the resin by transesterification, e.g., with methanol, followed by aminolysis or saponification. The protected peptide may be purified by silica gel chromatography.

The side chain protecting groups may be removed from the peptide by treating the aminolysis product with, for example, anhydrous liquid hydrogen fluoride in the presence of anisole or other carbonium ion scavenger, treatment with hydrogen fluoride/pyridine complex, treatment with tris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction with hydrogen and palladium on carbon or polyvinylpyrrolidone, or by reduction with sodium in liquid ammonia, preferably with liquid hydrogen fluoride and anisole at a temperature between about -10° and +10° C., preferably at about 0° C., for between about 15 minutes and 2 hours, preferably about 1.5 hours.

For peptides on the benzhydrylamine resin, the resin cleavage and deprotection steps may be combined in a single step utilizing liquid hydrogen fluoride and anisole as described above.

The solution may be desalted (e.g. with BioRad AG-3® anion exchange resin) and the peptide purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin in the acetate form; hydrophobic adsorption chromatography on underivatized co-poly(styrene-divinylbenzene), Amberlite® XAD; silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on Sephadex® G-25; countercurrent distribution; or high performance liquid chromatography (HPLC), especially reversed-phase HPLC on octyl- or octadecylsilylsilica (ODS) bonded phase column packing.

Thus, another aspect of the present invention relates to processes for preparing polypeptides and pharmaceutically acceptable salts thereof which processes comprise sequentially condensing protected amino acids on a suitable resin support, removing the protecting groups and resin support, and purifying the product, to afford analogs of the physiologically active truncated homologs and analogs of PTH and PTHrp, preferably of PTH(1-34) and PTHrp(1-34), in which the amino acids at positions (22-31) form an amphipathic α-helical peptide sequence, as defined above.

Recombinant Synthesis of the Polypeptides

Alternatively, the polypeptides of this invention may be prepared by cloning and expression of a gene encoding for the desired polypeptide. In this process, a plasmid containing the desired DNA sequence is prepared and inserted into an appropriate host microorganism, typically a bacteria, such as E. coli, or a yeast, such as Saccharomyces cerevisiae, inducing the host microorganism to produce multiple copies of the plasmid, and so of the cDNA encoding for the polypeptide analogs of this invention.

First, a synthetic gene coding for the selected PTH or PTHrp analog is designed with convenient restriction enzyme cleavage sites to facilitate subsequent alterations. Polymerase chain reaction (PCR), as taught by Mullis in U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporated herein by reference, may be used to amplify the sequence.

The amplified synthetic gene may be isolated and ligated to a suitable plasmid, such as a Trp LE plasmid, into which four copies of the gene may be inserted in tandem. Preparation of Trp LE plasmids is described in U.S. Pat. No. 4,738,921 and European Patent Publication No. 0212532, incorporated herein by reference. Trp LE plasmids generally produce 8-10 times more protein than Trp E plasmids. The multi-copy gene may then be expressed in an appropriate host, such as E. coli or S. cerevisiae.

The specific expression vector used herein was Trp LE 18 Prot (Ile³, Pro⁵) containing the following elements: a pBR322 fragment (EcoRI--BamHI) containing the ampicillin resistant gene and the plasmid origin of replication; an EcoRI-SacII fragment containing the trp promoter and the trpE gene; an HIV protease (Ile³,Pro⁵) gene fragment (SacII--HindIII); a bGRF gene fragment (HindIII--BamHI); and a transcription terminator from E. coli rpoc gene. The HIV protease and bGRF gene fragments are not critical and may be replaced with other coding sequences, if desired.

The expressed multimeric fusion proteins accumulate intracellularly into stable inclusion bodies and may be separated by centrifugation from the rest of the cellular protein. The isolated fusion protein is converted to the monomeric PTH or PTHrp analog and may be purified by cation exchange and/or reverse phase HPLC.

Alternative methods of cloning, amplification, expression, and purification will be apparent to the skilled artisan. Representative methods are disclosed in Maniatis, et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989), incorporated herein by reference.

Utility and Administration

The polypeptides of this invention are useful for the prevention and treatment of a variety of mammalian conditions manifested by loss of bone mass. In particular, the compounds of this invention are indicated for the prophylaxis and therapeutic treatment of osteoporosis and osteopenia in humans.

In general, the polypeptides of this invention, or salts thereof, are administered in amounts between about 0.01 and 1 μg/kg body weight per day, preferably from about 0.07 to about 0.2 μg/kg body weight per day. For a 50 kg human female subject, the daily dose of active ingredient is from about 0.5 to about 50 μgs, preferably from about 3.5 to about 10 μgs. In other meals, such as horses, dogs, and cattle, higher doses may be required. This dosage may be delivered in a conventional pharmaceutical composition by a single administration, by multiple applications, or via controlled release, as needed to achieve the most effective results, preferably one or more times daily by injection.

The selection of the exact dose and composition and the most appropriate delivery regimen will be influenced by, inter alia, the pharmacological properties of the selected polypeptide, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient.

Representative delivery regimens include oral, parenteral (including subcutaneous, intramuscular and intravenous), rectal, buccal (including sublingual), transdermal, and intranasal.

Pharmaceutically acceptable salts retain the desired biological activity of the parent polypeptide without toxic side effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonic acids, polygalacturonic acid and the like; (b) base addition salts formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cation formed from N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), e.g., a zinc tannate salt and the like.

A further aspect of the present invention relates to pharmaceutical compositions comprising as an active ingredient a polypeptide of the present invention, or pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable, non-toxic carrier. As mentioned above, such compositions may be prepared for parenteral (subcutaneous, intramuscular or intravenous) administration, particularly in the form of liquid solutions or suspensions; for oral or buccal administration, particularly in the form of tablets or capsules; for intranasal administration, particularly in the form of powders, nasal drops or aerosols; and for rectal or transdermal administration.

The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., (1985), incorporated herein by reference. Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. For oral administration, the formulation can be enhanced by the addition of bile salts or acylcarnitines. Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered spray. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.

When formulated for nasal administration, the absorption across the nasal mucous membrane may be enhanced by surfactant acids, such as for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cyclodextrins and the like in an amount in the range between about 0.2 and 15 weight percent, preferably between about 0.5 and 4 weight percent, most preferably about 2 weight percent.

Delivery of the compounds of the present invention to the subject over prolonged periods of time, for example, for periods of one week to one year, may be accomplished by a single administration of a controlled release system containing sufficient active ingredient for the desired release period. Various controlled release systems, such as monolithic or reservoir-type microcapsules, depot implants, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoretic devices and alternative injectable dosage forms may be utilized for this purpose. Localization at the site to which delivery of the active ingredient is desired is an additional feature of some controlled release devices, which may prove beneficial in the treatment of certain disorders.

One form of controlled release formulation contains the polypeptide or its salt dispersed or encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as copoly(lactic/glycolic) acid, as described in the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No. 4,675,189, incorporated by reference herein. The compounds or, preferably, their relatively insoluble salts, may also be formulated in cholesterol or other lipid matrix pellets, or silastomer matrix implants. Additional slow release, depot implant or injectable formulations will be apparent to the skilled artisan. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New York, 1978, and R. W. Baker, Controlled Release of Biologically Active Agents, John Wiley & Sons, New York, 1987, incorporated by reference herein.

The following specific Examples are intended to illustrate the synthesis and testing of representative compounds of the invention and should not be construed as limiting the scope of the claims. In the Examples, "m.p." is melting point, " α!_(D) ²⁵ " is the optical activity at 25° C. at the given concentration in the indicated solvent, "FAB" is fast atom bombardment mass spectrometry, and "AAA" is amino acid analysis, with expected values in parentheses following the observed values. The amino acid analyses were conducted on a Hewlett-Packard AminoQuant Analyzer following the manufacturer's recommended protocols. Primary amino acids were derivatized with o-phthalaldehyde; secondary amino acids with Fmoc. Fluorescent detection of the derivatized amino acids was used for quantification. The protected amino acids were obtained from Applied Biosystems Inc. (Foster City, Calif.).

EXAMPLE I

Compound 1 (SEQ ID NO:5) was prepared on 0.5 mmol scale by the solid phase method on 4-methylbenzhydrylamine resin, using an automated Applied Biosystems Model 430A peptide synthesizer. The α-amino groups were protected with t-butoxycarbonyl (Boc). The side chain protecting groups were: benzyl (Bzl) for Asp, Glu, and Ser; tosyl (Tos) for Arg; benzyloxymethyl (Bom) for His; and 2-chlorobenzyl (Cl-z) for Lys. The amino acids were coupled sequentially using N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt) following Stewart and Young (supra). After each amino acid coupling, the peptide was acetylated using a mixture of acetic anhydride and diisopropylethylamine in N-methylpyrrolidone. The completed peptide was cleaved from the resin with simultaneous deprotection of the side chain protecting groups using anhydrous HF (25 mL) in the presence of anisole (2.5 mL) at -10° C. for 30 minutes and at 0° C. for 60 minutes. After evaporation of the HF in vacuo, the residue was washed with anhydrous ether, and the crude peptide extracted with 10% acetic acid. Lyophilization of the 10% acetic acid extract gave 900 mgs of crude product. The peptide was purified by medium pressure ODS reversed phase column chromatography using a gradient of 22-45% CH₃ CN in 0.1% TFA. The product eluted in three fractions, which were concentrated and lyophilized to give 130 mgs of white solid of >98% purity.

Compound 1 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA--NH₂ (SEQ ID NO: 7)

Physical Data: m.p. 150°-159° C. α!_(D) ²⁵ -34.88° (c 0.16, H₂ O) FAB (C₁₇₅ H₃₀₀ N₅₆ O₅₁): M+H!⁺ 4005.5 AAA: Asp, 1.9(2); Glu, 5.6(6); Ser, 1.6(2); His, 2.7(3); Gly, 1.0 (1); Thr, 0.9 (1); Ala, 1.9 (2); Arg, 2.8 (3); Val, 1.0(1); Ile, 0.9(1); Leu, 7.3(8); Lys, 4.0(4).

Similarly, Compounds 2 and 5-18 were prepared and characterized, substituting PAM resin as needed for the synthesis of hydroxy-terminal polypeptides.

Compound 2 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA--OH (SEQ ID NO:6)

Physical Data: m.p. 154°-170° C. α!_(D) ²⁵ -49.35° (c 0.46 H₂ O) FAB (C₁₇₅ H₃₀₁ N₅₇ O₅₀): M+H!⁺ 4005.0 AAA: Asp, 2.1(2); Glu, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly 1.1(1); Thr, 1.0(1); Ala, 1.9(2); Arg, 3.0(3); Val, 1.2(1); Ile, 1.0(1); Leu, 7.8(8); Lys, 4.2(4).

Compound 5 AVSEHQLLHDKGKSIQDLRRRELLERLLERLHTA--OH (SEQ ID NO:15)

Physical Data: m.p. 147°-165° C. α!D²⁵ -49.17° (c 0.66 H₂ O) FAB (C₁₇₅ H₂₉₉ N₅₉ O₅₂): M+H!⁺ 4061 AAA: Asp, 2.1(2); Gly, 6.1(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 5.0(5); Val, 1.0 (1); Ile, 0.9 (1); Leu, 7.7 (8); Lys, 1.9 (2).

Compound 6 AVSEHQLLHDRGRSIQDLRRRELLERLLERLHTA--OH (SEQ ID NO: 16)

Physical Data: m.p. 150°-1700° C. α!_(D) ²⁵ -48.65° (c 0.54, H₂ O) FAB (C₁₇₅ H₂₉₉ N₆₃ O₅₂): M+H!⁺ 4118.0 AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.2(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 6.9(7); Val, 1.0(1); Ile, 1.0(1); Leu, 7.8(8).

Compound 7 AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA--OH (SEQ ID NO: 17)

Physical Data: m.p. 177°-182° C. α!_(D) ²⁵ -46.17° (c 0.14, H₂ O) FAB (C₁₇₆ H₃₀₄ N₆₄ O₅₀): M+H!⁺ 4117 AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 6.7(7); Val, 1.0(1); Ile, 1.0(1); Lys, 7.7(8); Lys, 0.9(1).

Compound 8 AVSEHQLLHDKGKSIQDLRRRELLEKLLRKLHTA--OH (SEQ ID NO: 5)

Physical Data: m.p. 147°-165° C. α!_(D) ²⁵ -49.17° (c 0.66 H₂ O) FAB (C₁₇₆ H₃₀₅ N₅₉ O₄₉): M+H!⁺ 4033.0 AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 2.7(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 2.0(2); Arg, 3.9(4); Val, 1.0 (1); Ile, 1.0 (1); Leu, 7.9 (8); Lys, 4.0 (4).

Compound 9 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTAGRR--OH (SEQ ID NO:10)

Physical Data: m.p. 158°-160° C. α!_(D) ²⁵ -44.76° (c 0.1, H₂ O) FAB (C₁₈₉ H₃₂₆ N₆₄ O₅₅): M!⁺ 4375.0 AAA: Asp, 2.0(2); Glu, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly, 2.3 (2); Thr, 1.0 (1); Ala, 1.9 (2); Arg, 5.0 (5); Val, 1.2(1); Ile, 1.0(1); Leu, 7.8(8); Lys, 4.3(4).

Compound 10 AVSEAQLLHDLGKSIQDLRRRELLEKLLEKLHAL--OH (SEQ ID NO:14)

Physical Data: m.p. 170°-175° C. α!_(D) ²⁵ -31.59° (c 0.54, H₂ O) FAB (C₁₇₄ H₃₀₀ N₅₂ O₅₁): M+H!⁺ 3936.0 AAA: Asp, 2.0(2); Glu, 6.0(6); Ser, 1.8(2); His, 2.0(2); Gly, 1.2(1); Ala, 3.0(3); Arg, 2.8(3); Val, 1.1(1); Ile, 1.0(1); Leu, 9.9(10); Lys, 3.0(3).

Compound 11 AVSEHQLLHDKGKSIQDLRRRELLEKLLELLKEL--NH₂ (SEQ ID NO:11)

Physical Data: m.p. 172°-174° C. α!_(D) ²⁵ -43.29° (c 0.2, H₂ O) FAB (C₁₇₉ H₃₁₁ N₅₅ O₅₂): M+H!⁺ 4065.8 AAA: Asp, 2.2(2); Glu, 7.7(7); Ser, 1.7(2); His, 2.0(2); Gly, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1) Ile, 1.0(1); Leu, 9.3(9); Lys, 5.1(5).

Compound 12 AVSEIQFXHNLGKHLSSXERVELLEKLLEKLHNY--NH₂ (X=Nle, SEQ ID NO:23)

Physical Data: m.p. 178° C. α!_(D) ²⁵ -36.88° (c 0.4, H₂ O) FAB (C₁₈₂ H₂₉₅ N₅₀ O₅₁): M+H!⁺ 4001.6 Asp, 2.1(2); Glu, 6.5(6); Ser, 2.7(3); His, 3.1(3); Gly, 1.1(1); Ala, 1.0(1); Arg, 1.0(1); Tyr, 0.8(1); Val, 2.0(2); Phe, 1.0(1); Ile, 0.9(1); Leu+Nle, 8.5(7+2); Lye, 3.1(3).

Compound 13 AVSEIQFXHNLGKHLSSXRRRELLEKLLEKLHNY--NH₂ (X=Nle, SEQ ID NO:24)

Physical Data: m.p. 260° C. α!_(D) ²⁵ -37.02° (c 0.2, H₂ O) FAB (C₁₈₄ H₃₀₄ N₅₆ O₄₉): M+H!⁺ 4084 AAA: Asp, 2.1(2); Glu, 5.5(5); Ser, 2.6(3); His, 3.1(3); Ala, 1.0(1); Gly, 1.1(1); Arg, 3.2(3); Tyr, 1.0(1); Val, 1.0(1); Phe, 1.0(1); Ile, 1.0(1); Leu, 9.0(9); Lys, 3.0 (3).

Compound 14 AVSEHQLLHDKGKSIQDLRRRALAEALAEALHTA--NH₂ (SEQ ID NO:20)

Physical Data: m.p. 190°-225° C. α!_(D) ²⁵ -56.58° (c 0.36 H₂ O) FAB (C₁₆₁ H₂₇₂ N₅₄ O₄₉): M+H!⁺ 3747.0 AAA: Asp, 2.1(2); Glu, 4.9(5); Ser, 1.7(2); His, 2.6(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 7.6(7); Arg, 2.8(3); Val, 1.2(1); Ile, 1.0(1); Leu, 6.6(6); Lys, 1.9(2).

Compound 15 AVSEHQLLHDKGKSIQDLARRELLEKLLEKLHTA--NH₂ (SEQ ID NO:12)

Physical Data: m.p. 170°-180° C. α!_(D) ²⁵ -48.19° (c 0.2, H₂ O) FAB (C₁₇₂ H₂₉₃ N₅₃ O₅₁): M+H!⁺ 3919.0 AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.7(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu, 8.0(8); Lys, 4.4(4).

Compound 16 AVSEHQLLHDKGKSIQDLRRAELLEKLLEKLHTA--NH₂ (SEQ ID NO:13)

Physical Data: m.p. 190°-195° C. α!_(D) ²⁵ -50.50° (c 0.4 H₂ O) FAB (C₁₇₂ H₂₉₃ N₅₃ O₅₁): M+H!⁺ 3919.0 AAA: Asp, 2.1(2); Glu, 6.0(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu, 7.5(8); Lys, 4.2(4).

Compound 17 AVSEHQLLHDKGKSIQDLRRRSLLSSLLSSLHTA--NH₂ (SEQ ID NO:21)

Physical Data: m.p. 195°-204° C. α!_(D) ²⁵ -67.11° (c 0.3 H₂ O) FAB (C₁₆₃ H₂₈₀ N₅₄ O₅₀): M+H!⁺ 3796.0 AAA: Asp, 2.1(2); Glu, 2.9(3); Ser, 6.8(7); His, 3.1(3); Gly, 1.2(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Val, 1.0 (1); Ile, 1.0 (1); Leu, 8.2 (8); Lys, 2.0 (2).

Compound 18 AVSEHQLLHDKGKSIQDLRRRAFYDKVAEKLHTA--NH₂ (SEQ ID NO:22)

Physical Data: m.p. 200°-207° C. α!_(D) ²⁵ -60.26° (C 0.6, H₂ O) FAB (C₁₇₄ H₂₈₄ N₅₆ O₅₀): M+H!⁺ 3960.0 AAA: Asp, 2.9(3), Glu, 3.5(4); Ser, 1.4(2); His, 2.6(3); Gly, 0.9(1); Thr, 1.0(1); Ala, 4.0(4); Arg, 3.0(3); Tyr, 0.9(1); Val, 1.9(2); Phe, 1.1(1); Ile, 0.9(1); Leu, 3.6(4); Lys, 4.1(4).

EXAMPLE II

Met³⁴, Ala³⁵ ! Compound 1, AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTMA-NH₂, (SEQ ID NO:25), was prepared and purified following the procedures of Example I. This polypeptide was converted to the homoserine lactone as follows. The purified peptide (160 mgs) was dissolved in 44% formic acid (4 mL). This solution was combined with a premixed solution of cyanogen bromide (700 mgs) and phenol (1.6 mgs) in 44% formic acid (4 mL) at 0° C. The solution was stirred at 0° C. for 2 hr and at room temperature for 2 hrs. The formation of the product was monitored by HPLC (Vydac® C-18, 300 Å, 4.6×250 mm, flow of 1.2 mL/min, gradient 25-45% acetonitrile in 0.1% TFA over 10 min). The reaction was complete within 4 hr. Half of the sample was concentrated and purified by preparative RP-HPLC (Vydac® C-18, gradient 25-45% acetonitrile in 0.1% TFA). The homoserine lactone peptide fractions were pooled and lyophilized to give 28 mgs of white solid of >95% purity, Compound 4.

Compound 4 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX (X=hSerlac, SEQ ID NO:9)

Physical Data: m.p. 138°-142° C. α!_(D) ²⁵ -50.66° (c 0.1, H₂ O) FAB (C₁₇₆ H₂₉₉ N₅₅ O₅₂): M+H!⁺ 4017.61 AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.0(3); Thi, 1.1(1); Ala, 1.1(1); Arg, 2.7(3); Val, 1.0(1); Ile, 1.0(1); Leu, 8.2(8); Lys, 3.8(4); Gly 1.09(1); hSer, 1.09 (1).

EXAMPLE III

To prepare the homoserine amide, the crude hSerlactone analog, Compound 4, was concentrated and treated with 25 mL saturated NH₃ in methanol. The solution was stirred at 0° C. for 2 hr and at room temperature for 16 hr. The reaction was monitored by HPLC (Vydac® C-18, 300 Å, 4.6×250 mm, flow of 1.2 mL/min, gradient 20-45% acetonitrile in 0.1% TFA) and was complete within 18 hr. The solution was concentrated and purified by preparative RP-HPLC (Vydac® C-18, gradient of 25-45% acetonitrile in 0.1% TFA). The homoserine amide peptide fractions were pooled and lyophilized to give 30 mgs of white solid of >98% purity, Compound 3.

Compound 3 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX--NH₂ (X=hSer, SEQ ID NO:8)

Physical Data: m.p. 138°-142° C. α!_(D) ²⁵ -45.97° (C 0.25, H₂ O) FAB (C₁₇₆ H₃₀₂ N₅₆ O₅₂): M+H!⁺ 4033.9 AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.6(2); His, 2.8(3); Gly, 0.97(1); hSer, 0.97(1); Thi, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Val, 1.0(1); Ile, 1.0(1); Leu, 7.6(8); Lys, 3.9 (4).

EXAMPLE IV

Following Example I, the protected peptide-resin BocAVS(Bzl)E(OBz)H(Bom)QLLHD(OBzl)R(Ts)GR(Ts)S(Bzl)IQD(OBz)LR(Ts)R(Ts)E(OBz)LLE(OBzl)R(Ts)LLK(Fmoc)R(Ts)LH(Bom)T(Bzl)A--O--PAM was synthesized on a 0.35 mmol scale. All N.sup.α groups were protected with t-butoxycarbonyl (Boc); side chain protecting groups were as indicated. After completion of the synthesis, the peptide resin was treated with 50 mL of 20% piperidine in dimethylformamide (DMF) at room temperature for 30 minutes to remove the fluorenylmethoxycarbonyl (Fmoc) protecting group on lysine. The resin was washed successively with DMF, MeOH, CH₂ Cl₂ and dried to give 1.6 g partially protected peptide. 0.8 g (0.175 mmol) of the partially protected peptide was acylated on lysine with 0.44 g (0.3 mmol) of methoxydi(ethyleneoxy) acetic acid PEG(2)CH₂ COOH! in the presence of 0.16 g (0.3 mmol) benzotriazolyloxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBop) and 0.067 (0.525 mmol) of diisopropylethyl amine (DIEA) in 20 mL DMF at room temperature for 5 hrs. After 5 hrs., the resin was filtered and washed successively with DMF, MeOH and CH₂ Cl₂. The acylation step was repeated twice until negative ninhydrin result on the resin was obtained. The final peptide was cleaved from the resin with removal of the side chain protecting groups and purification as in Example I; 100 mgs of Compound 19 were obtained.

Compound 19 AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA--OH (SEQ ID NO:18)

    CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C═O

Physical Data: m.p. 145°-195° C. α!_(D) ²⁵ -44.60° (c 0 2 H₂ O) FAB (C₁₈₃ H₃₁₆ N₆₄ O₅₄): M+H!⁺ 4276.2 AAA: Asp, 2.1(2); Glu, 5.0(5); Ser, 1.6(2); His, 2.9(3); Gly, 0.9(1); Thr, 1.9(2); Arg, 7.1(7); Val, 1.1(1); Ile, 1.0(1); Leu, 8.0(8); Lys, 0.9(1).

EXAMPLE V

Peptide was synthesized, cleaved and purified in the same manner as in Example IV except 2-methoxypoly(ethyleneoxy) acetic acid PEG(5000)CH₂ CO₂ H! was used as the acylating agent. 0.8 g (0.175 mmol) of partially protected peptide yielded 300 mgs of pure Compound 20.

Compound 20 AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA--OH (SEQ ID NO:19)

    CH.sub.3 O (CH.sub.2 CH.sub.2 O).sub.110 CH.sub.2 C═O

Physical Data: m.p. 105° C. α!_(D) ²⁵ -22.95° (c 0.11, 50% aq. HOAc) AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.6(2); His, 2.6(3); Gly, 1.1(1); Thr, 1.1(1); Arg, 7.3(7); Val, 0.8(1); Ile, 0.9(1); Leu, 8.3(8); Lys, 1.1(1); Ala, 1.8(2).

EXAMPLE VI Synthesis of hPTHrp(1-34) analog gene

A synthetic gene coding for the hPTHrp(1-34) analog Compound 4 (SEQ ID NO:9) was designed having the nucleotide sequence and enzyme restriction sites shown in FIG. 1.

The requisite oligodeoxynucleotides were prepared with a DNA synthesizer (Milligen/Biosearch) using the phosphoramidite process of Sinha, et al., Nucleic Acid Research 12, 4539-4557 (1984), incorporated herein by reference. After deprotection, the crude oligonucleotides were purified by gel electrophoresis on preparative 15% polyacrylamide gels. The oligonucleotides were located with UV, excised from the gel, desalted over Waters c18 Sep-pak® cartridges, and lyophilized.

Amplification Via polymerase chain reaction (PCR) was carried out on a Perkin-Elmer Cetus thermal cycler, with 25 cycles of: 94° C. for 1 minute, 50° C. for 2 minutes, and 72° C. for 3 minutes, using reagents, including Taq polymerase, from the "GeneAmp" DNA amplification kit (Perkin-Elmer Cetus).

Two overlapping oligonucleotides, an 88mer (2 μg), PTH3 (SEQ ID NO:31): ##STR17## and an anti-sense 90mer (2 μg), PTH4 (SEQ ID NO:32): ##STR18## were prepared as the template DNA sequence for the hPTHrp (1-34) analog gene. Utilizing the two flanking primers, PTHPCR1: CCTCTAGATC TCCGCGGCGC TAG (SEQ ID NO:33) and PTHPCR2: CCTCGAAGCT TATGCATCAT TATC (SEQ ID NO:34), the entire gene was amplified by PCR. The amplified DNA products were purified by gel electrophoresis on 4% NuSieve® agarose gel. The band containing the synthetic hPTHrp(1-34) analog gene, approximately 150 bases in length, was excised from the gel and approximately 200 ng of DNA isolated by Elu-Quik® glass gel DNA extraction (Schleicher & Schuell, Keene, N.H.).

EXAMPLE VII Molecular Cloning of an hPTHrp(1-34)1 Analog Gene

To subclone the hPTHrp(1-34) analog gene of Example VI, 200 ng of the amplified DNA was isolated and cut by restriction enzymes HinD III and Sac II. As shown in FIG. 2, the DNA was ligated to 2 μg TrpLE 18 Prot (Ile³, Pro⁵) plasmid previously cleaved with Hind III and Sac II.

The resulting plasmid, TrpLE 18 hPTHrp(1-34)1, containing one copy of the hPTHrp(1-34) analog gene was then transformed into competent E. coli HB 101 cells (CLONTECH, Palo Alto, Calif.). Transformants were subjected to PCR analysis to verify insertion. Transformed cell colonies were selected and boiled in 200 μL of water for 5 minutes; 2 μL were subjected to PCR with two primers flanking the insert. The PCR product was then analyzed on 1% agarose gel to confirm the presence of one copy of the hPTHrp(1-34) gene insert. TrpLE 18 hPTHrp(1-34)1 construct was then verified by DNA sequencing on an automated DNA sequencer (Applied Biosystems Model 373A, Foster City, Calif.) using the vendor's Dye Deoxy Terminator Sequencing kit.

EXAMPLE VIII Construction of a Trp LE 18 Vector Containing Multiple Copies of the hPTHrp(1-34) Analog Gene

Unique Nhe I and Xba I restriction sites are located near the beginning and end of the hPTHrp(1-34) analog gene sequence. These two sites, which recognize different sequences, but produce identical single strand cohesive termini, allow the construction of multiple copy hPTHrp (1-34) genes within the Trp LE 18 vector.

The strategy for constructing repeated hPTHrp(1-34) sequences in tandem is outlined in FIG. 3. In separate reactions, 5 μg of plasmid Trp LE 18 hPTHrp(1-34)1 containing a single copy of the gene was cleaved with Ban Hi+Nhe I and Xba I+Bam HI. From each digest, about 300 ng of the fragment containing the hPTHrp(1-34) analog gene was isolated. These two fragments were mixed and ligated to form the Trp LE 18 hPTHrp(1-34)2 plasmid. This plasmid was used to transform competent E. coli HB 101 cells. Sizing of the transformed PCR products on 1% agarose gel was used to determine the presence of two copies of the hPTHrp(1-34) gene insert. TrpLE 18 hPTHrp(1-34)2 containing two copies of the gene was then confirmed by DNA sequencing. The correct fusion of the two hPTHrp(1-34) genes results in the elimination of Nhe I and Xba I sites at the junction. This makes the remaining Xba I and Nhe I sites flanking the tandem genes unique.

By repeating this process the final plasmid Trp LE 18 hPTHrp(1-34)4 containing four copies of the hPTHrp(1-34) gene was constructed, as shown in FIG. 4. The sequence of Trp LE 18 hPTHrp(1-34)4 was found to be correct by DNA sequence analysis.

EXAMPLE IX Expression and Purification of Trp LE 18 hPTHrp(1-34)4

Induction of the Trp LE 18 hPTHrp(1-34)4. A starter culture of 50 mL of LB culture media, J. H. Miller, "Experiments in Molecular Genetics," p.431 (1972), incorporated herein by reference, containing 50 μg/mL ampicillin and 100 μg/mL tryptophan, was inoculated with E. coli cells containing Trp LE 18 hPTHrp(1-34)4 plasmid, and grown overnight at 37° C. with vigorous shaking to an A₅₅₀ of about 6. Two liters of LB culture media for production were pre-warmed to 37° C. and seeded with 20 mL of the starter culture to give an A₅₅₀ of about 0.06. The culture was then grown with vigorous shaking to an A₅₅₀ of between 0.6 and 0.8, whereupon 2 mL of a 10 mg/mL solution of indole acrylic acid (IAA) was added. Growth was continued with good aeration for about 16 hr to a final A₅₅₀ of about 6 (typically, between 4 and 10). The cells were concentrated by centrifugation and resuspended in 500 mL of 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA buffer solution (Tris buffer).

The suspension was sonicated using a Heat Systems-Ultrasonics, Inc. model 220F sonicator (equipped with a 3/4" horn) operated at 50% of full capacity to avoid overheating.

To determine the extent of induction, the whole cells were analyzed by SDS-PAGE. The gene products derived from the TrpLE 18 hPTHrp(1-34)4 construct were seen as a major band of the predicted MW of approximately 17,000. This accounts for as much as 10% of the total cellular protein.

Isolation of the Fusion Protein. The cell lysate was centrifuged for 15 min. at about 3600×g to pellet the Trp LE 18 hPTHrp(1-34)4 fusion protein; the supernatant was discarded. The pellet was resuspended in 200 mL Tris buffer. (typically 40-80 A₅₅₀ /mL).

EXAMPLE X Processing of the Fusion Protein and Purification of homo-Serlactone hPTHrp(1-34) peptide

Cleavage of the methionine residues flanking the hPTHrp(1-34) multimeric fusion protein with CNBr releases the desired homo-Serlactone hPTHrp(1-34) polypeptide, which was purified as described below.

CNBr Treatment of Fusion Protein. The washed pellet of TrpLE 18 hPTHrp(1-34)4 fusion protein was resuspended by gently stirring in 60 mL of 70% formic acid (about 20 mg/mL total protein; typically material from 1000 A₅₅₀ units of cells is dissolved in 3 mL). A few drops of octanol were added and N₂ bubbled through the solution for 20 minutes before adding 5.5 g CNBr. This reaction was allowed to proceed for 6 hours at 25° C. before an equal volume of 50:50 MeOH:H₂ O was mixed with the sample and subsequently removed by rotary evaporation. After 2 to 4 repetitions of this process, the bulk of the formic acid and CNBr were essentially removed. The sample was then evaporated to dryness, redissolved in 200 mL water and lyophilized for storage.

Purification of homo-Serlactone hPTHrp(1-34). The CNBr cleaved supernatant was dialyzed against 50 mM KH₂ PO₄ pH 6.5 for 24 hours with multiple changes. During dialysis, pH was maintained at 6.5. After dialysis, the precipitates were removed by high speed centrifugation. The supernatant was clarified through a Gelman 0.45μ filter device (Acrodisc 4184).

Cation Exchange Chromatography. Initial purification was accomplished by cation exchange chromatography on a Bio-Gel TSK-SP-5PW HPLC column (21.5×150 mm). Chromatographic conditions for a flow rate of 8 mL/min and a yield of approximately 12 mg of highly purified homo-Serlactone hPTHrp(1-34) peptide were:

1. Column equilibration in 50 mM KH₂ PO₄, pH 6.5

2. Load 10 mL clarified supernatant (approximately 1.5 L culture broth or 2.4 g inclusion).

3. Wash column with 50 mM KH₂ PO₄ pH 6.5 containing 50 mM NaCl until baseline is stabilized.

4. Elute column with 50 mM KH₂ PO₄ pH 6.5 containing 90 mM NaCl. Collect fractions for about 45 minutes.

5. Analyze the 90 mM NaCl fractions for homo-Serlactone hPTHrp(1-34) content by C18 HPLC before pooling and storage.

Reverse Phase HPLC Chromatography. A reverse phase Poros R/H 4.6×100 mm column (Perseptive Biosystems, Cambridge, Mass.) was used for the final purification step. The chromatographic conditions were as follows:

    ______________________________________                                         Mobile phase A:                                                                             0.1% trifluoroacetic acid (TFA)/water                             B:           0.1% trifluoroacetic acid (TFA)/CH.sub.3 CN                       TIME            FLOW     % B                                                   ______________________________________                                           0 min         4 ml/min 15                                                    5.0 min         4 ml/min 40                                                    5.2 min         4 ml/min 100                                                   6.8 min         4 ml/min 100                                                   7.0 min         4 ml/min 15                                                    ______________________________________                                    

Retention time of the homo-Serlactone hPTHrp(1-34), Compound 4, was approximately 2.943 minutes. The purified peptide was approximately 98% pure as determined by mass spectroscopy.

EXAMPLE XI

The compounds of this invention were evaluated for their effect on bone mass in ovariectomized rats, generally in accord with the procedures of Gunness-Hey and Hock, Metab. Bone Dis. 5:177 181 (1984), incorporated by reference herein.

Adult Sprague-Dawley female rats were acclimatized, weight grouped (n=9, 10 or 12), and subjected to bilateral ovariectomy (OVX) or sham surgery. Dosing was initiated 17 days after surgery and continued for 20 days. Test compound was administered subcutaneously once a day in 2% rat serum/saline vehicle.

After 20 days of dosing, the rats were sacrificed and the right femurs excised. The femurs were cut in half and the distal half femurs (DHF) were further separated into trabecular bone (TB) and cortical bone (CB) by drilling out the trabeculae. Calcium was extracted and measured by Calcette calcium analyzer and expressed as mean bone Ca in mg/DHF/100 g body weight.

The two sample t-test was used to compare OVX and sham groups. One-way ANOVA was used to compare OVX groups, followed by Fisher's LSD multiple comparison to compare each treatment group to vehicle.

Ovariectomy induced substantial total bone loss, primarily from trabecular bone. Total bone calcium was 47 to 54% lower than for sham-operated controls.

bPTH(1-34) and hPTHrp(1-34) at 80 μg/kg/day provided statistically significant increases in total bone calcium for treated OVX rats, ranging from 18 to 53%; however, there was no significant increase in cortical bone calcium.

Compounds of this invention, dosed at 80 μg/kg/day, increased total bone calcium by from 66 to 138% and trabecular calcium by from 87 to 128%. Cortical bone calcium, trabecular thickness, and bone volume were also significantly increased over untreated OVX controls.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 34                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsn                               151015                                                                         SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                               202530                                                                         AsnPhe                                                                         (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        AlaValSerGluIleGlnPheMetHisAsnLeuGlyLysHisLeuSer                               151015                                                                         SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                               202530                                                                         AsnPhe                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuSer                               151015                                                                         SerLeuGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                               202530                                                                         AsnPhe                                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgPhePheLeuHisHisLeuIleAlaGluIleHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuArgLysLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 34                                                               (D) OTHER INFORMATION: /note="Xaa34 =homoserine"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         ThrXaa                                                                         (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 34                                                               (D) OTHER INFORMATION: /note="Xaa34 =homoserine lactone"                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         ThrXaa                                                                         (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         ThrAlaGlyArgArg                                                                35                                                                             (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuLys                               202530                                                                         GluLeu                                                                         (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuAlaArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgAlaGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       AlaValSerGluAlaGlnLeuLeuHisAspLeuGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         AlaLeu                                                                         (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluArgLeuLeuLysArgLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 29                                                               (D) OTHER INFORMATION: /note="Xaa29 =                                          lysine- (OCCH2(OCH2CH2)2OCH3)"                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluArgLeuLeuXaaArgLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 29                                                               (D) OTHER INFORMATION: /note="Xaa29 =                                          lysine- (OCCH2(OCH2CH2)110OCH3"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluArgLeuLeuXaaArgLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgAlaLeuAlaGluAlaLeuAlaGluAlaLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgSerLeuLeuSerSerLeuLeuSerSerLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgAlaPheTyrAspLysValAlaGluLysLeuHis                               202530                                                                         ThrAla                                                                         (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 8                                                                (D) OTHER INFORMATION: /note="Xaa8 =norleucine"                                (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 18                                                               (D) OTHER INFORMATION: /note="Xaa18 =norleucine"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       AlaValSerGluIleGlnPheXaaHisAsnLeuGlyLysHisLeuSer                               151015                                                                         SerXaaGluArgValGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         AsnTyr                                                                         (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 8                                                                (D) OTHER INFORMATION: /note="Xaa8 =norleucine"                                (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 18                                                               (D) OTHER INFORMATION: /note="Xaa18 =norleucine"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       AlaValSerGluIleGlnPheXaaHisAsnLeuGlyLysHisLeuSer                               151015                                                                         SerXaaArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         AsnTyr                                                                         (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: N-terminal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                               151015                                                                         AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                               202530                                                                         ThrMetAla                                                                      35                                                                             (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: internal                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 8                                                                (D) OTHER INFORMATION: /note="Xaa8 =glutamic acid or                           arginine"                                                                      (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                           (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: /note="Sequence 26 is embedded at                       positions 22 to 31 of sequences 5, 6, 7, 8, 9, 10,                             11, 12, 13, and 14."                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       GluLeuLeuGluLysLeuLeuXaaLysLeu                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: internal                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                    (B) LOCATION: 8                                                                (D) OTHER INFORMATION: /note="Xaa8 =glutamic acid,                             lysine, or lysine-(OCCH2PEGX)"                                                 (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                           (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: /note="Sequence 27 is embedded at                       positions 22 to 31 of sequences 15, 16, 17, 18,                                and 19. "                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       GluLeuLeuGluArgLeuLeuXaaArgLeu                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: internal                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: /note="Sequence 28 is embedded at                       positions 22 to 31 of sequence 20."                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       AlaLeuAlaGluAlaLeuAlaGluAlaLeu                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: internal                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: /note="Sequence 29 is embedded at                       positions 22 to 31 of sequence 21."                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       SerLeuLeuSerSerLeuLeuSerSerLeu                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: internal                                                    (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: /note="Sequence 30 is embedded at                       positions 22 to 31 of sequence 22."                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       AlaPheTyrAspLysValAlaGluLysLeu                                                 1510                                                                           (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 88 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       CCTCTAGATCTCCGCGGCGCTAGCATGGCTGTTTCTGAACATCAGCTGCTTCATGACAAA60                 GGTAAATCGATTCAAGATCTGAGACGTC88                                                 (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 90 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: YES                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       CCTCGAAGCTTATGCATCATTATCTAGACATAGTATGCAGCTTTTCAAGCAGTTTCTCCA60                 GCAGCTCGCGACGTCTCAGATCTTGAATCG90                                               (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       CCTCTAGATCTCCGCGCGCTAGC23                                                      (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: YES                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       CCTCGAAGCTTATGCATCATTATC24                                                     __________________________________________________________________________ 

We claim:
 1. A process for the synthesis of a modified parathyroid hormone related polypeptide (PTHrP) of the formula:Ala Val Ser Glu Xaa⁵ Gln Leu Leu His Asp Xaa¹¹ Gly Xaa¹³ Ser Ile Gln Asp Leu Xaa¹⁹ Arg Xaa²¹ Xaa²²⁻³¹ Xaa³² Xaa³³ Xaa³⁴ Term, wherein:Xaa⁵ is His or Ala; Xaa¹¹ and Xaa¹³ are independently Lys, Arg, or Leu; Xaa¹⁹ and Xaa²¹ are independently Ala or Arg; Xaa²²⁻³¹ is selected from the group consisting of (SEQ ID NOS: 26, 27, 28, 29, and 30); Xaa³² is His or Lys; Xaa³³ is Thr, Glu, or Ala; Xaa³⁴ is Ala, hSer, Tyr, or Leu; and Term is OH, NR₂, lactone, or Gly Arg Arg, where R is H or (C₁ -C₄) alkyl; and the pharmaceutically acceptable salts thereof, which process comprises expressing a nucleic acid sequence encoding said polypeptide.
 2. A process of claim 1 wherein said polypeptide comprises a polypeptide wherein Xaa²²⁻³¹ is (SEQ ID NO:26).
 3. A process of claim 2 wherein said polypeptide is selected from the group consisting of (SEQ ID NOS: 7, 8 and 9).
 4. A process of claim 1 wherein said polypeptide comprises a polypeptide wherein Xaa²²⁻³¹ is (SEQ ID NO:27).
 5. A process of claim 1 wherein said polypeptide comprises a polypeptide wherein Xaa²²⁻³¹ is (SEQ ID NO:28).
 6. A process of claim 1 wherein said polypeptide comprises a polypeptide wherein Xaa²²⁻³¹ is (SEQ ID NO:29).
 7. A process of claim 1 wherein said polypeptide comprises a polypeptide wherein Xaa²²⁻³¹ is (SEQ ID NO:30).
 8. A process for the synthesis of a modified parathyroid hormone (PTH) polypeptide of the formula:Xaa¹ Val Ser Glu Ile Gln Xaa⁷ Xaa⁸ His Asn Leu Gly Lys His Leu Xaa¹⁶ Ser Xaa¹⁸ Xaa¹⁹ Arg Xaa²¹ Xaa²²⁻³¹ His Ash Xaa³⁴ Term, wherein:Xaa¹ is Ser or Ala; Xaa⁷ is Leu or Phe; Xaa⁸ is Met or Nle; Xaa¹⁶ is Ash or Ser; Xaa¹⁸ is Leu, Met, or Nle; Xaa¹⁹ is Glu or Arg; Xaa²¹ is Val or Arg; Xaa²²⁻³¹ is selected from (SEQ ID NOS: 26, 27, 28, 29, or 30); Xaa³⁴ is Phe or Tyr; and Term is OH or NR₂, where R is H or a (C₁ -C₄)alkyl; and the pharmaceutically acceptable salts thereof, which process comprises expressing a nucleic acid sequence encoding said polypeptide.
 9. An expression vector comprising a nucleic acid sequence encoding a PTHrP analog of the formula:Ala Val Ser Glu Xaa⁵ Gln Leu Leu His Asp Xaa¹¹ Gly Xaa¹³ Ser Ile Gln Asp Leu Xaa¹⁹ Arg Xaa²¹ Xaa²²⁻³¹ Xaa³² Xaa³³ Xaa³⁴ Term, wherein:Xaa⁵ is His or Ala; Xaa¹¹ and Xaa¹³ are independently Lys, Arg, or Leu; Xaa¹⁹ and Xaa²¹ are independently Ala or Arg; Xaa²²⁻³¹ is selected from the group consisting of (SEQ ID NOS: 26, 27, 28, 29, and 30); Xaa³² is His or Lys; Xaa³³ is Thr, Glu, or Ala; Xaa³⁴ is Ala, hSer, Tyr, or Leu; and Term is OH, NR₂, lactone, or Gly Arg Arg, where R is H or (C₁ -C₄) alkyl.
 10. A host cell transformed with an expression vector of claim
 9. 11. An expression vector encoding a modified parathyroid hormone (PTH) polypeptide of the formula:Xaa¹ Val Ser Glu Ile Gln Xaa⁷ Xaa⁸ His Asn Leu Gly Lys His Leu Xaa¹⁶ Ser Xaa¹⁸ Xaa¹⁹ Arg Xaa²¹ Xaa²²⁻³¹ His Asn Xaa³⁴ Term, wherein:Xaa¹ is Ser or Ala; Xaa⁷ is Leu or Phe; Xaa⁸ is Met or Nle; Xaa¹⁶ is Asn or Set; Xaa¹⁸ is Leu, Met, or Nle; Xaa¹⁹ is Glu or Arg; Xaa²¹ is Val or Arg; Xaa²²⁻³¹ is selected from (SEQ ID NOS: 26, 27, 28, 29, or 30); Xaa³⁴ is Phe or Tyr; and Term is OH or NR₂, where R is H or a (C₁ -C₄)alkyl.
 12. A host cell transformed with an expression vector of claim
 11. 13. The polypeptide of SEQ ID NO:
 26. 14. The polypeptide of SEQ ID NO:
 27. 15. The polypeptide of SEQ ID NO:
 28. 16. The polypeptide of SEQ ID NO:
 29. 17. The polypeptide of SEQ ID NO:
 30. 